| BackgroundTraumatic brain injury(TBI)is a serious trauma caused by external force on the head.It can be divided into two categories: closed injury and open injury,The mortality rate is between 4% and 7%,The mortality rate of severe brain injury is as high as 50%-60%.Excessive glutamate release has been implicated as a major contributor to multiple secondary damage to post-traumatic brain injury(TBI),including neurodegeneration.Prior to the presence of behavior change,TBI leads to degeneration and apoptosis of peripheral cortical neurons,which is believed to be relevant to inappropriately increased extracellular glutamate concentration and glutamatergic receptor activation.Acutely promoting brain glutamate clearance with a blood-based scavenging system,glutamate oxaloacetate transaminase(GOT),reduces apoptosis in peripheral cortical neurons.Overall,the present findings support the importance of clearance of glutamate post-TBI and provide new evidence of the mechanism of glutamate-induced apoptosis which leads to a development of secondary damage to post-TBI.ObjectiveTo investigate the neuroprotective effect and mechanism of Blood-based glutamate scavenger rGOT after TBI.Methods1.Rat TBI model was produced by free fall in a stereotaxic frame,adult male SD rats were randomly divided into four groups: vehicle group,GOT group,oxa group,GOT+oxa group.2.Establish a microdialysis-perfusion system to collect the right hippocampus ISF,right ventricle CSF,high pressure liquid chromatography(HPLC)glutamate levels in the sample.3.Brain tissue samples were collected in post-TBI,determine the total glutamate concentration in the hippocampus and cerebral cortex by using a glutamate assay kit.Western blot was used to detect the changes of EAAT1 and EAAT2 in the brain tissues in each group.4.Use a glutamate assay kit determine serum glutamate concentration.5.TUNEL observes and evaluates neuronal apoptosis,calculates apoptosis rate.Results1.Repeated systemic treatment with blood-based glutamate scavengers decreased the transient elevation of glutamate level in hippocampus interstitial fluid: In the control animals that were treated with repeated rGOT s.c.,the glutamate level of hippocampus ISF was not significantly affected(6.0 ± 2.0 ?M at day 1,7.7 ± 1.1 ?M at day 2,5.8 ± 1.6 ?M at day 3,and 6.4 ± 1.4 ?M at day 4,P > 0.05 compared with 7.9 ± 0.6 ?M at day 0,n = 4)as well as the control group treated with vehicle of GOT s.c.(6.1 ± 0.6 ?M at day 1,6.6 ± 1.0 ?M at day 2,5.5 ± 0.4 ?M at day 3,and 9.3 ± 1.0 ?M at day 4,P > 0.05 compared with 7.5±1.2 ?M at day 0,n =4,P > 0.05 compared with counterpart time points in GOT-treated group).Importantly,in the animals that received TBI induction,a transient glutamate enhancement was observed at day 1 in hippocampus ISF,while the glutamate level returned to baseline from day 2(24.1 ± 1.9 ?M at day 1,P <0.05 compared with 7.9±0.6?M at day 0,5.2 ± 1.9 ?M at day 2,6.3 ± 1.6 ?M at day 3,and 5.0 ± 1.2 ?M at day 4,P >0.05 when compared each time point apart from day 1,n =4).And the repeated s.c.injection of rGOT at a 12-h interval abrogated the transient glutamate shoot out on day 1 post-TBI(9.8 ± 1.9 ?M at day 1,P < 0.05 compared with counterpart time point in TBI animals that received vehicle treatment,P > 0.05 when compared 5.7 ± 0.7 ?M at day 0,7.8 ± 1.1 ?M at day 2,5.0±0.5?M at day 3,and 7.0±0.9?M at day 4,n =4).2.Repeated systemic treatment with blood-based glutamate scavengers induced a delayed glutamate clearance in ventricular CSF: We further investigated the time frame of repeated treatment with blood-based glutamate scavengers affecting the glutamate level in ventricular CSF samples.However,unlike the data we gathered from hippocampus ISF,the glutamate level in ventricular CSF increased gradually post the induction of TBI,and reached a peak on day 3(12.1±1.7?M at day 1,19.4 ± 0.2 ?M at day 2,21.0 ± 2.7 ?M at day 3,and 13.9 ± 1.8 ?M at day 4,P <0.05 compared with 5.9 ± 0.4 ?M at day 0,n = 4).In the animals that received repeatedly applied rGOT for 4 days post-TBI,the glutamate level in ventricular CSF did not significantly decreased until day 4 post-TBI(12.7 ± 1.6 ?M at day 1,16.8 ± 2.1 ?M at day 2,and 17.7 ± 4.8 ?M at day 3,P <0.05 compared with 4.9±0.9?M at day 0,P >0.05 compared with counterpart time points in vehicle-treated group,7.0 ± 0.9 ?M at day 4,P >0.05 compared with base-line level on day 0,P < 0.05 compared with day 4 in vehicle-treated group,n =4).The repeated application of either blood-based glutamate scavengers(3.8 ± 0.6 ?M at day 1,2.5 ± 0.1 ?M at day 2,3.0±0.3?M at day 3,and 4.2 ± 1.3 ?M at day 4,P >0.05 compared with 3.8 ± 0.6 ?M at day 0,n = 4)or its vehicle(4.6 ±1.5 ?M at day 1,2.3 ± 0.2 ?M at day 2,5.2 ± 0.3 ?M at day 3,and 3.0 ± 0.6 ?M at day 4,P > 0.05 compared with 3.6 ±0.5 ?M at day 0,n = 4)did not significantly affect the glutamate level in ventricular CSF.3.Repeated systemic treatment with blood-based glutamate scavengers reduced apoptosis in peripheral cortical neurons.On the first day and the fourth day after TBI modeling in rats,neuronal apoptosis was evaluated and the apoptosis rate was calculated.(The apoptotic rate was 6.4%±1.6% on the first day in the sham group,59.5%±10.1% in the TBI group,and 38%±7.8% in the treatment intervention group.The TBI group was compared with the sham operation group,The apoptosis rate was significantly increased,and subcutaneous injection of GOT significantly reduced the apoptosis of peripheral cortical neurons on day 1 after TBI,P<0.05).ConclusionThe application of GOT reduced apoptosis in peripheral cortical neurons by decreasing glutamate level in hippocampus ISF in vivo. |
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